During FY15 we accomplished the following: 1) Continued studies with DeltaE&#956; mice. In this strain E&#956; is replaced by TetO and Gal4 binding sites. Flow cytometric analyses demonstrated that these mice recapitulate the phenotype of E&#956; deficiency. We are ready to commence protein recruitment studies to investigate function of individual, and combinations of, transcription factors that activate E&#956;. 2) &#956;E1 mutated mice that were generated by gene targeting in ES cells were bred to delete the Neo gene from the locus, and then crossed with RAG2-deficient mice to maintain IgH in unrearranged configuration. This strain will be ready for experiments in 6 to 8 weeks. 3) We generated a second &#956;E1 mutation using CRISPR/Cas9. This strain will be on a C57Bl6 background, and will complement the results obtained with &#956;E1 mutation created in ES cells of 129 strain background. 4) We generated &#956;E2+&#956;E5 mutated IgH alleles using CRISPR/Cas9. These mice are being bred for preliminary phenotyping. 5) We generated &#956;A+&#956;B mutated IgH alleles using CRISPR/Cas9. These mice are being bred for preliminary phenotyping. Cumulatively, studies with &#956;E1, &#956;E2+&#956;E5 and &#956;A+&#956;B mutated IgH alleles will provide the first comprehensive analysis of the function of individual motifs in a complex mammalian enhancer. 6) We studied the epigenetic and transcriptional state of IgH alleles in CD4+ CD8+ (DP) thymocytes. IgH alleles undergo partial rearrangements in DP cells, the basis for which remains a mystery. We hypothesized that the nuclear milieu of DP cells may be deficient in a subset of factors required for complete activation of IgH alleles, and thereby serve as a natural mutation for the identification of such factors. We made two striking observations. First, the VH domain does not undergo CTCF-dependent compaction in DP cells despite normal levels of CTCF binding. Second, anti-sense transcripts arising at E&#956; are substantially reduced in DP cells. We are investigating the molecular bases for these differences.